Silymarin, an extract of milk thistle seeds, prevents liver injury and disease progression in many animal models. Recent clinical studies indicate that silymarin also reduces hepatitis C viral load and progression of hepatitis C liver disease in humans. If we are to understand and exploit the full value of this natural product, we must understand how silymarin protects the liver, which has not been clearly elucidated. In our NCCAM-funded R21, we discovered that silymarin blocks hepatitis C virus (HCV) infection and have since defined the stages of the HCV lifecycle that are blocked by silymarin. We have isolated and evaluated the 8 major components of silymarin in hepatoprotection assays that measure antiviral, antioxidant, and anti-inflammatory functions. This proposal will use two parallel approaches to discover the mechanisms of action of silymarin. Specifically, we will identify the physiological target(s) of silymarin components that confer hepatoprotection in the forms of antiviral, antioxidant, and anti-inflammatory activities. Our hypothesis is that silymarin components interact with mammalian biomolecules in a specific and productive manner to cause changes in signal transduction and gene expression in a cell to protect the liver. We will address the hypothesis in two specific aims that will 1) identify transcriptional changes using microarrays of liver cell lines and primary hepatocyte cultures treated with silymarin and silymarin-derived pure compounds, 2) identify and validate cellular targets of silymarin compounds using chemical proteomics. By examining silymarin-induced gene regulation and elucidating cellular targets of silymarin, this application is intentionally responsive to RFA-AT-11-001. At the end of the funding period, we anticipate that we will know the cellular targets of silymarin and how silymarin causes changes in a cell at a systems biology level, thereby providing the first detailed explanation of how silymarin protects the liver. The novel data emanating from this research project are expected to pave the way for identification of biomarkers of silymarin treatment and efficacy, as well as guiding refinements in silymarin- based natural product treatments for liver disease.